The liver displays inherent tolerogenicity and liver allografts promote tolerance more readily than other organs. Compared to circulating or secondary lymphoid tissue DC, liver myeloid (m) DC produce lower IL-12, secrete more IL-10, induce alloAg-specific T cell hyporesponsiveness and prolong allograft survival. Liver DC are refractory to Toll-like receptor (TLR) agonism and nuclear factor (NF)?B activation,- a critical determinant of DC maturation. Underlying mechanisms may contribute to their regulatory function and the inherent tolerogenicity of hepatic allografts. Importantly, our new data show that selective deletion of donor mDC prevents 'spontaneous' liver transplant tolerance in mice. Mechanisms underlying unresponsiveness to TLR agonism in DC in general, and in liver DC, in particular, are poorly defined. We have identified the signaling adaptor DNAX-activating protein of 12KDa (DAP12), that can mediate inhibition of TLR activation, and that is upregulated in liver mDC, as a critical novel regulator of liver transplant tolerance. We also show that intrahepatic IL-6/ signal transducer and activator of transcription (STAT3) signaling inhibits liver mDC maturation and function. Consequently, we hypothesize that negative regulators of TLR signaling, such as IRAK-M (that we show is upregulated with DAP12 in liver mDC) confer resistance to maturation and tolerogenic capacity. We further postulate that TGF and IL-10, produced in the liver, potentiate liver DC tolerogenicity through molecular 'crosstalk' between their respective signaling pathways (smad for TGF, and STAT3 for IL-6 and IL-10) and the TLR signaling pathway. The tolerogenic function attributed to liver mDC may contribute to induction/maintenance of liver transplant tolerance in the face of continuous TLR agonism and other pro-inflammatory stimuli. Our studies will provide new mechanistic insight into the molecular regulation of liver DC function and its impact on alloreactive T cell responses and liver transplant tolerance. We will also evaluate the therapeutic potential of negative regulators of TLR signaling/liver DC maturation for restoration/promotion of transplant tolerance. We propose the following aims: AIM 1: Elucidate the role of inducible regulators of TLR signaling in development of mouse liver mDC tolerogenicity; AIM 2: Evaluate the role of endogenous anti-inflammatory mediators and transplant-related pro-inflammatory factors in acquisition of tolerogenic function by liver mDC; AIM 3: Establish using an innovative in vivo approach, the roles of donor (versus host) mDC, TLR4, and negative regulators of TLR signaling/DC activation in control of T cell responses and tolerance to liver allografts.